Distinct cortical and sub-cortical neurogenic …...ORIGINAL ARTICLE Distinct cortical and...

ORIGINAL ARTICLE

Distinct cortical and sub-cortical neurogenic domainsfor GABAergic interneuron precursor transcription factorsNKX2.1, OLIG2 and COUP-TFII in early fetal humantelencephalon

Ayman Alzu’bi1,2• Susan Lindsay2

• Janet Kerwin2• Shi Jie Looi1,2

•

Fareha Khalil1,2• Gavin J. Clowry1

Received: 18 May 2016 / Accepted: 18 November 2016 / Published online: 30 November 2016

� The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract The extent of similarities and differences

between cortical GABAergic interneuron generation in

rodent and primate telencephalon remains contentious. We

examined expression of three interneuron precursor tran-

scription factors, alongside other markers, using immuno-

histochemistry on 8–12 post-conceptional weeks (PCW)

human telencephalon sections. NKX2.1, OLIG2, and

COUP-TFII expression occupied distinct (although over-

lapping) neurogenic domains which extended into the

cortex and revealed three CGE compartments: lateral,

medial, and ventral. NKX2.1 expression was very largely

confined to the MGE, medial CGE, and ventral septum

confirming that, at this developmental stage, interneuron

generation from NKX2.1? precursors closely resembles

the process observed in rodents. OLIG2 immunoreactivity

was observed in GABAergic cells of the proliferative zones

of the MGE and septum, but not necessarily co-expressed

with NKX2.1, and OLIG2 expression was also extensively

seen in the LGE, CGE, and cortex. At 8 PCW,

OLIG2? cells were only present in the medial and anterior

cortical wall suggesting a migratory pathway for

interneuron precursors via the septum into the medial

cortex. By 12 PCW, OLIG2? cells were present through-

out the cortex and many were actively dividing but without

co-expressing cortical progenitor markers. Dividing

COUP-TFII? progenitor cells were localized to ventral

CGE as previously described but were also numerous in

adjacent ventral cortex; in both the cases, COUP-TFII was

co-expressed with PAX6 in proliferative zones and TBR1

or calretinin in post-mitotic cortical neurons. Thus COUP-

TFII? progenitors gave rise to pyramidal cells, but also

interneurons which not only migrated posteriorly into the

cortex from ventral CGE but also anteriorly via the LGE.

Keywords Ganglionic eminences � Inhibitory

interneurons � Neurodevelopment � Neuronal fate

specification � Pallium � Subpallium

Abbreviations

COUP-TFII Chicken ovalbumin upstream promotor-

transcription factor 2

DLX2 Distal-less homeobox 2

NKX2.1 NK2 homeobox 1

OLIG2 Oligodendrocyte lineage transcription

factor 2

PAX6 Paired box 6

TBR1 and

TBR2

T-box brain 1 and 2

CGE Caudal ganglionic eminence

LGE Lateral ganglionic eminence

MGE Medial ganglionic eminence

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00429-016-1343-5) contains supplementarymaterial, which is available to authorized users.

& Susan Lindsay

[email protected]

& Gavin J. Clowry

[email protected]

1 Institute of Neuroscience, Newcastle University, Framlington

Place, Newcastle upon Tyne NE2 4HH, UK

2 Institute of Genetic Medicine, Newcastle University,

International Centre for Life, Parkway Drive,

Newcastle upon Tyne NE1 3BZ, UK

123

Brain Struct Funct (2017) 222:2309–2328

DOI 10.1007/s00429-016-1343-5

http://orcid.org/0000-0002-6489-8329

http://dx.doi.org/10.1007/s00429-016-1343-5

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Introduction

Humans have considerably expanded cognitive abilities

compared to all other species which may be dependent on the

evolution of a greater interconnectedness of a larger number

of functional modules (DeFelipe 2011; Buckner and Krienen

2013). This not only depends on the physical presence of

neurons, axon pathways, and synapses, but also on syn-

chronicity of neural activity between cortical areas binding

together outputs of all neurons within a spatially distributed

functional network (Singer and Gray 1995; Fries 2009). The

synchronicity essential to higher order processing is depen-

dent on the activity of gamma-aminobutyric acidergic

(GABAergic) interneurons (Whittington et al. 2011; Buzsaki

and Wang 2012), and we might predict a more sophisticated

functional repertoire for interneurons in higher species

(Ballesteros-Yanez et al. 2005; Molnar et al. 2008; DeFelipe

2011; Povysheva et al. 2013; Clowry 2015).

Is this expanded repertoire of functional types matched

by an evolution of their developmental origins? It is well

established in rodents that GABAergic interneurons are

born almost entirely outside the neocortex in the ganglionic

eminences and associated structures (such as the preoptic

area) from which they migrate tangentially into the cortex

(De Carlos et al. 1996; Parnavelas 2000; Marın and

Rubenstein 2001; Welagen and Anderson 2011). The

ganglionic eminences are divided into three main neuro-

genic domains: the lateral, medial, and caudal ganglionic

eminences (LGE, MGE, and CGE, respectively). The LGE

is the origin of the striatal projection neurons and a small

population of olfactory bulb interneurons (Waclaw et al.

2009). The MGE and the CGE are the major sites of cor-

tical interneurogenesis (Xu et al. 2004; Butt et al. 2005).

Recent studies support the idea that generation of

interneurons in the ventral telencephalon may be more

complicated in primates, which have evolved a large and

complex outer subventricular zone in the ganglionic emi-

nences (Hansen et al. 2013). In addition, proportionally,

more interneurons appear to be produced in the CGE, the

majority of which populate the superficial layers of the

cortex (Hansen et al. 2013; Ma et al. 2013). Whether or not

the cortical proliferative zones are a source of interneuro-

genesis, to what extent and significance, is a contentious

issue (Molnar and Butt 2013; Clowry 2015). Some

researchers have proposed that primates generate

interneurons in the proliferative zones of the cortex (Le-

tinic et al. 2002; Petanjek et al. 2009; Zecevic et al. 2011;

Radonjic et al. 2014a; Al-Jaberi et al. 2015) as well as in

the ganglionic eminences. Other groups have convincingly

argued that interneuronogenesis is essentially the same in

primates as in rodent models (Hansen et al. 2013; Ma et al.

2013; Arshad et al. 2016). As there is growing evidence

that conditions, such as autism, schizophrenia, and con-

genital epilepsy, may have developmental origins in the

failure of interneuron production and migration (De Felipe

1999; Lewis et al. 2005; Uhlhaas and Singer 2010; Marın

2012), it is important that we understand fully the simi-

larities and differences between human development and

that in our animal models.

Therefore, we have carried out a detailed study of expres-

sion of three transcription factors expressed by interneuron

progenitors, NKX2.1, OLIG2, and COUP-TFII. We looked

between the ages of 8–12 post-conceptional weeks (PCW)

which have been a relatively neglected period of development

in the previous studies of interneurogenesis. NKX2.1 is con-

sidered the key regulator of MGE-derived GABAergic

interneuron specification (Sussel et al. 1999; Xu et al. 2004;

Butt et al. 2008; Du et al. 2008) and the only transcription

factor that distinguishes the MGE from other subcortical

domains, including in the human embryo at 7 PCW (Pauly

et al. 2014). In mice, following the early loss of Nkx2.1

function, the MGE acquires an LGE-like molecular specifi-

cation (Sussel et al. 1999), whereas the late conditional loss of

function switches the MGE to CGE in character (Butt et al.

2008). In rodents, the MGE is the major source of cortical

GABAergic interneurons and Nkx2.1 expression in this

region is required for the specification of somatostatin and

parvalbumin expressing interneurons from MGE progenitors

(Xu et al. 2004; Butt et al. 2008; Du et al. 2008).

In the rodent, Olig2 expressing progenitors in the MGE

give rise to GABAergic interneurons at an earlier stage of

development and oligodendrocytes at later stages (Miyoshi

et al. 2007). In the human, OLIG2 has been detected prin-

cipally in the proliferative zones of the ganglionic eminences

between 5–15 PCW, prior to significant expression of

markers for oligodendrocyte precursors, with a spread into

the cortex by 20 gestational weeks, along with co-expression

of OLIG2 with markers for immature neurons, neurogenic

radial glia, and intermediate progenitor cells (Jakovceski and

Zecevic 2005; Jackocevski et al. 2009).

COUP-TFII, in rodents, is preferentially expressed in

the CGE as well as in interneurons migrating into the

cortex (Kanatani et al. 2008; Lodato et al. 2011). CGE-

derived interneurons migrate caudally to the most posterior

part of the telencephalon (Yozu et al. 2005; Faux et al.

2012), and COUP-TFII is essential to establish this caudal

migratory stream (Kanatani et al. 2008). Reinchisi et al.

(2012) have provided some evidence that COUP-TFII may

play a similar role in human forebrain development.

However, the precise roles of COUP-TFs in specifying the

CGE-derived interneurons are still unclear.

Expression of the transcription factor PAX6 was also

investigated alongside the three GABAergic markers.

PAX6 is considered a marker for dorsal telencephalic

2310 Brain Struct Funct (2017) 222:2309–2328

123

radial glia giving rise to glutamatergic neurons in rodents

(Hevner et al. 2006), and in human, PAX6 expression

delineates the cortical ventricular and subventricular pro-

liferative zones (Bayatti et al. 2008a). However, in humans,

it is also known to be expressed in a gradient across the

proliferative layers of the LGE revealing its boundary with

the MGE (Pauly et al. 2014; Harkin et al. 2016).

The present study aimed to map and quantify the

expression of the three interneuron progenitor markers in

the human telencephalon at an important stage of devel-

opment prior to the arrival of thalamic innervation to

delineate the extent of cortical interneurogenesis. This aim

was achieved, demonstrating distinct neurogenic domains

for all three including expression in the developing cortex.

In addition, careful observation of these expression patterns

revealed a complex organization for the CGE and provided

evidence for anterior and medial migration pathways for

interneuron precursors into the cortex from the ganglionic

eminences in addition to the more generally recognised

lateral and posterior pathways.

Methods and materials

Human tissue

Human fetal tissue from terminated pregnancies was

obtained from the joint MRC/Wellcome Trust-funded

Human Developmental Biology Resource (HDBR, http://

www.hdbr.org; Gerrelli et al. 2015). All tissues were col-

lected with appropriate maternal consent and approval

from the Newcastle and North Tyneside NHS Health

Authority Joint Ethics Committee. Fetal samples ranging in

age from 8 to 12 PCW were used. Ages were estimated

from the measurements of foot and heel to knee length

compared with the fetal staging chart as described by Hern

(1984). Brains were isolated and fixed for at least 24 h at

4 �C in 4% paraformaldehyde dissolved in 0.1 M phos-

phate-buffered saline (PBS) (PFA; Sigma Aldrich). Once

fixed, whole or half brains (divided sagittally) were dehy-

drated in a series of graded ethanols before embedding in

paraffin. Eight brain samples were cut at 8 lm section

thickness in three different planes; horizontally, sagittally,

and coronally, mounted on slides and used for haema-

toxylin and eosin staining (H&E) and immunostaining.

Immunoperoxidase histochemistry

This was carried out according to previously described

protocols (Harkin et al. 2016). Briefly, paraffin sections

were dewaxed by treatment with xylene and rehydrated via

three changes of graded ethanol/water mixes. Endogenous

peroxidase activity was blocked by treatment with

methanol peroxide for 10 min. Sections were rinsed in tap

water and boiled by microwave treatment in 10 mM citrate

buffer pH 6 for antigen retrieval for 10 min. Sections were

then incubated with the appropriate normal blocking serum

in Tris buffered saline (TBS) before incubation with the

primary antibody (diluted in 10% normal blocking serum)

overnight at 4 �C. Details of all the primary antibodies

used in this study are found in Table 1. Then, sections were

washed and incubated with the biotinylated secondary

antibody for 30 min at room temperature (Vector Labora-

tories Ltd., Peterborough, UK) at 1:500 dilution in 10%

normal serum in TBS followed by washing and incubation

with avidin-peroxidase for 30 min (ABC-HRP, Vector

Labs). The sections were developed with diaminobenzidine

(DAB) solution for 10 min (Vector Labs) washed, dehy-

drated, and mounted using DPX (Sigma-Aldrich, Poole,

UK).

Immunofluorescence (double and triple labelling)

We used a novel immunofluorescent staining method,

Tyramide Signal Amplification (TSA), that permits

sequential double and triple staining using antibodies from

the same species without cross-reactions (Goto et al. 2015;

Harkin et al. 2016). Sections were treated as described

above until the secondary antibody stage, then they were

incubated with HRP-conjugated secondary antibody for

30 min [ImmPRESSTM HRP IgG (Peroxidase) Polymer

Detection Kit, Vector Labs], washed twice for 5 min in

TBS, and incubated in dark for 10 min with fluorescein

tyramide diluted at 1/500 in 19 Amplification buffer

(Tyramide Signal Amplification (TSATM) fluorescein plus

system reagent, Perkin Elmer, Buckingham, UK). Tyra-

mide reacts with HRP to leave fluorescent tags covalently

bound to the section.

Prior to starting the second round of staining, sections

were first washed in TBS and boiled in 10 mM citrate

buffer to remove all antibodies and unbound fluorescein

from the first round. Sections were then incubated in 10%

normal serum before incubating with the second primary

antibody for 2 h at room temperature. Following washing,

sections were again incubated with ready to use HRP-

conjugated secondary antibody and then incubated with

CY3 tyramide for 10 min [Tyramide Signal Amplification

(TSATM) CY3 plus system reagent, Perkin Elmer]. The

same steps were repeated for the third round of staining (if

triple labelling was needed) using CY5 Tyramide (Tyra-

mide Signal Amplification (TSATM) CY5 plus system

reagent, Perkin Elmer). Sections were washed before

applying 40,6-diamidino-2-phenylindole dihydrochloride

(DAPI; Thermo Fisher Scientific, Cramlington, UK) and

mounted using Vectashield Hardset Mounting Medium

(Vector Labs).

Brain Struct Funct (2017) 222:2309–2328 2311

123

http://www.hdbr.org

http://www.hdbr.org

Imaging

All immunoperoxidase staining figures presented in this

study were captured using a Leica slide scanner and Zeiss

Axioplan 2 microscope. The double immunofluorescent

figures were obtained with a Zeiss Axioimager Z2 apotome

and Triple Immunofluorescent images were obtained with a

Nikon A1R confocal microscope. Processing of images,

which included only adjustment of brightness and sharp-

ness, was achieved using the Adobe Photoshop CS6

software.

Quantification

Nine sections from a 12 PCW embryo were selected at

intervals along the anterior–posterior axis and

immunoperoxidase stained for NKX2.1, OLIG2, or COUP-

TFII. For each section, using images obtained with the

slide scanner, a counting box 100 lm-wide and approx.

750 lm-deep (the exact dimensions were recorded and

used in calculations) was placed over the ventricular and

subventricular zones (VZ and SVZ), with the 100 lm edge

parallel to the ventricular surface, at either two or three

locations within the following regions of the section (if

present); lateral, dorsal, medial, or ventral cortex, MGE,

LGE, or ventral CGE, or sub-cortical septum. The number

of immunopositive cells within these counting boxes was

recorded and the area of the box measured. From these

counts, the average density of immunopositive cells in the

VZ/SVZ of each anatomical region was recorded. Using a

3D reconstruction of a 12–13 PCW fetal brain made from

MRI scans (available at http://database.hudsen.eu), we

calculated the volume of each of the brain regions that we

had counted cells in and then multiplied the volume of the

brain region by the average density of immunopositive

cells in that region to give an estimate of the total number

of immunopositive cells in that brain region (see Table 2).

In this way, we took into account that although the cortex

contained a low density of some cell types, the much larger

volume of the cortical regions might contain a relatively

large number of cells.

Results

Examination of our immunoperoxidase labelled sections

for various markers at low magnification revealed details of

the characteristics of the CGE and septum in human not

fully reported on before in detail. Therefore, we have

begun the results section by describing these regions,

before moving on to describe the level of expression of

each GABAergic interneuron precursor transcription factor

in different parts of the telencephalon, aided by a more

detailed knowledge of CGE and septal sub-compartments.

The position and subdivisions of the caudal

ganglionic eminence (CGE)

The position of the CGE can be determined with respect to

other subcortical landmarks in H&E stained sections

(Suppl. Figure 1). For example, in the horizontal plane, at

the level of the internal capsule, the MGE and LGE

appeared as prominent bulges into the lateral ventricles,

and in a rostral position relative to the internal capsule. The

CGE can be seen as the part of the GE positioned caudally

to the internal capsule immediately adjacent to the ventral/

temporal cortex (Suppl. Figure 1a). In sagittal sections, the

most dorsal part of the CGE appeared as well-defined

protrusion into the lateral ventricle, close to the hip-

pocampus (Suppl. Figure 1b). The central part lies next to

the narrow ventral extension of the lateral ventricles

(Suppl. Figure 1c). In a coronal plane at the level of the

rostral half of thalamus, parts of MGE and LGE can be

seen dorsal to the internal capsule (Suppl. Figure 1d and e)

and only the most ventral part of the CGE was observed

ventral to the internal capsule and close to the ventral/

temporal cortex (Suppl. Figure 1e). At the level of the

caudal half of thalamus and caudal to the internal capsule,

Table 1 Primary antibodies

used in this studyPrimary antibody Species Dilution Supplier

KI67 Mouse monoclonal 1/150 Dako, Ely, UK

TBR1 Rabbit polyclonal 1/1000 Abcam, Cambridge, UK

TBR2 Rabbit polyclonal 1/200 Abcam

PAX6 Rabbit polyclonal 1/500 Cambridge Bioscience, Cambridge, UK

NKX2.1 Mouse monoclonal 1/150 Dako

COUPT-FII Mouse monoclonal 1/500 R&D Systems, Abingdon, UK

OLIG2 Rabbit polyclonal 1/1000 Merck Millipore, Watford, UK

CalR Mouse monoclonal 1/2000 Swant, Marly, Switzerland

GAD65/67 Rabbit polyclonal 1/200 Merck Millipore


123

http://database.hudsen.eu

the CGE was present but not the MGE and LGE (Suppl.

Figure 1f).

Immunohistochemical analysis revealed that the CGE

can be subdivided into three compartments, medial, lateral,

and ventral (Figs. 1, 2). PAX6 was expressed in a gradient

with higher expression in the cortical proliferative zones to

lower expression in the LGE. In addition to this gradient, a

well-defined cortical/subcortical boundary was also

revealed by an abrupt change in the expression pattern of

PAX6 located ventral to the physical sulcus between the

cortex and the bulge of LGE. Whereas in the cortex, PAX6

expression is confined to easily recognisable ventricular,

subventricular, and intermediate zones (VZ, SVZ, and IZ)

in the LGE; this organization was not well defined, with a

more diffuse cell population than in the subcortical SVZ

(Fig. 1a, c). A complementary expression pattern of PAX6

and NKX2.1 was seen across the GE, as previously

described at 7–8 PCW (Pauly et al. 2014). While PAX6

was expressed in the LGE, decreasing in expression from

the lateral boundary with the cortex to the boundary with

the MGE, NKX2.1 was almost exclusively expressed in the

MGE (Fig. 1a–d). A marked boundary between PAX6 and

NKX2.1 expression was located at the level of the

intereminential sulcus between LGE and MGE. This divi-

sion also extended continuously and caudally into the CGE.

PAX6 expression extended to the VZ of the most dorsal

and lateral part of the CGE which protruded into the lateral

ventricle, whereas NKX2.1 expression extended to the

medial part of the CGE which lay close to the ventral

extension of the lateral ventricles (Figs. 1c, d, 2a, a0, b, b0).The previous studies in rodents defined these two parts of

the CGE as caudal extensions of the LGE and MGE,

respectively (Corbin et al. 2003; Flames et al. 2007).

Accordingly, these two domains can be defined as ‘‘LGE-

like CGE’’ (LCGE) and ‘‘MGE-like CGE’’ (MCGE).

Interestingly, these two domains could not be distin-

guished in the most ventral part of CGE, which was located

immediately adjacent to the ventral/temporal cortex

(Figs. 1e, f, 2a00, b00). Thus, we propose that there is a third

compartment to the CGE in human, ‘‘ventral CGE’’

(VCGE), which was characterized by strong immunoreac-

tivity for PAX6, but not for NKX2.1. Despite the high

PAX6 expression in the VCGE, there was still a distinct

boundary between it and the adjacent cortex, characterized

by a thicker cortical VZ compared with more condensed

PAX6? cells in the SVZ of the VCGE (Fig. 2a00, b00). As

the CGE is recognised as the birth place of calretinin

(CalR) expressing interneurons in rodents (Nery et al.

2002; Butt et al. 2005), we studied the expression of CalR

in the three defined compartments of the CGE. CalR was

preferentially expressed in cells of the VZ and SVZ of the

LCGE and VCGE. Only scattered cells were observed in

MCGE. CalR immunostaining also revealed a distinct

boundary between the VCGE and the ventral cortex. In

VCGE, CalR was expressed in the VZ and SVZ, but in the

ventral cortex, expression of CalR was mainly confined to

the SVZ with only scattered CalR? cells in the VZ

(Fig. 2c, c0, c00).

Subdivisions of the septum

Similar to the ganglionic eminences, the septum is a

subcortical structure that exhibited complementary

expression of PAX6 and NKX2.1. The most ventral part

of septum could be defined as MGE-like septum char-

acterized by strong immunoreactivity for NKX2.1 but

not PAX6 expression. More dorsally, we found LGE-

like septum, which was characterized by moderate

expression of PAX6 but not NKX2.1. The most dorsal

part of septum had a cortical rather than sub-cortical

identity, manifested by higher PAX6 expression in the

VZ and SVZ and expression of TBR1 by post-mitotic

cells in the SVZ, IZ, and cortical plate (Fig. 1e–h;

Suppl. Figure 2).

Table 2 Cell counts in proliferative zones of 12 PCW fetus

Compartment Volume

(mm3)

NKX2.1 OLIG2 COUP-TFII

Density (cells/

mm3 9 103)

Number

(9106)

% Density (cells/

mm3 9 103)

Number

(9106)

% Density (cells/

mm3 9 103)

Number

(9106)

%

MGE 155.9 593.4 92.5 86.5 450.7 70.3 35.9 43.3 6.7 1.5

LGE 380.3 10.6 4.0 3.8 176.6 67.2 34.3 329.8 125.4 27.2

VCGE 161.6 5.5 0.9 0.8 191.1 30.9 15.8 1118.3 180.7 39.2

Septum 46.8 147.4 6.9 6.5 81.1 3.8 1.9 15.2 0.7 0.2

Cortex 2196.6 1.2 2.6 2.4 10.8 23.7 12.1 67.4 148 32.1

The volume of each compartment, which refers only to the SVZ and VZ, was estimated from a 3D reconstruction of post-mortem MRI scans.

Cell counts were made in randomly placed counting frames, a density calculated, and the value extrapolated to represent the whole compartment.

The percentage of cells refers to the proportion in the compartment of the total number of cells expressing each transcription factor in the

proliferative zones


123

Expression of NKX2.1 in human fetal telencephalon

8–12 PCW

NKX2.1 expression was almost entirely confined to the

MGE, including the MCGE, and the ventral part of the

septum (Fig. 1b, d, f, h; Suppl. Figure 2). We observed that

NKX2.1 immunoreactivity was strongly expressed in cells

of the VZ and SVZ of the MGE, and in cells probably

migrating through LGE, mostly within the non-prolifera-

tive mantle zone, toward the cortex (Fig. 3a) as has been

reported in many species (Corbin et al. 2001; Hansen et al.

2013; Quintana-Urzainqui et al. 2015). GAD65/67

immunoreactivity was expressed mainly in cells of the

SVZ of the MGE and LGE, but only scattered cells were

found in the VZ. In the LGE, GAD65/67 immunoreactivity

marked a clear cortical/subcortical boundary (Fig. 3a).

Double immunofluorescence labelling revealed that

NKX2.1 and GAD65/67 co-localized in the majority of

cells in MGE. GAD65/67 was also expressed in a few

migrating cells in the cortex; however, no NKX2.1? cells

were found in the cortex at 8 PCW (Fig. 3a) in agreement

with the previous findings in rodents and human that

NKX2.1 is downregulated in GABAergic interneurons

migrating out of MGE (Marın and Rubenstein 2001;

Letinic et al. 2002; Hansen et al. 2013).

However, at 12 PCW, we found scattered NKX2.1? cells

in the cortical VZ and SVZ sometimes far removed from the

ganglionic eminences and septum (Fig. 3b), but no NKX2.1

Fig. 1 Complementary expression of PAX6 and NKX2.1 in the

ganglionic eminences and septum of human fetal forebrain at 8 PCW.

a PAX6 was expressed in a gradient with higher expression in the

proliferative zone of the cortex to lower expression in the LGE and its

caudal extension (LCGE). b NKX2.1 expression was mainly confined

to the MGE and its caudal extension (MCGE). c, d Higher magni-

fication of boxed areas in a and b. e, f Ventral sections cut at the level

of the septum; PAX6 was densely expressed in the proliferative zone

of VCGE, but no NKX2.1 expression was found in VCGE. g, h Higher

magnification of boxed areas in e and f. Similar to the ganglionic

eminences, PAX6 was expressed in a gradient from the cortex part to

dorsal part of the septum (LGE-like septum) and NKX2.1 was

exclusively expressed in the most ventral part of the septum (MGE-

like septum). Scale bars 1 mm in f (and for a, b, and e); 100 lm in

h (and for c, d, and g). Ant anterior, Pos posterior


123

positive (NKX2.1?) cells were observed to co-express KI67

(not shown) a marker for active cell division (Scholzen and

Gerdes 2000). We next quantified the average density of

NKX2.1? in the proliferative zones of the MGE, septum,

LGE, VCGE, and the cortex of a 12PCW brain cut in the

coronal plane (Fig. 3c; Table 2), and estimated that 93% of

NKX2.1 cells in proliferative layers were found in the MGE

and ventral septum, 4.6% in the LGE and VCGE, and only

2.4% in the cortex.

Expression of OLIG2 in human fetal ventral

telencephalon 8–12 PCW

Distinct patterns of OLIG2 immunoreactivity were seen in

MGE, LGE, and CGE. At both 8 and 12 PCW, OLIG2 was

strongly expressed in cells of the VZ and SVZ of the MGE

(Figs. 4a, b, 5a–c), but weaker expression was observed in

the VZ and SVZ of the LGE (Fig. 4a, b). We observed

aggregations of OLIG2? cells amongst OLIG2- cells

throughout in the SVZ of the MGE (Fig. 5c). In CGE

compartments, both the level and the pattern of expression

of OLIG2 in the MGE and LGE were extended caudally to

the MCGE and LCGE, respectively (Fig. 4f–j); however,

only scattered OLIG2? cells were observed in VCGE

(Fig. 4k).

Since OLIG2 was highly expressed in the NKX2.1-ex-

pressing neurogenic domain (MGE and ventral septum),

we examined the cellular co-localization of these two

markers in the MGE at 8 and 12 PCW. Interestingly, we

found three population of cells, NKX2.1-/OLIG2? ,

Fig. 2 Subdivisions of the CGE in sagittal sections at 12 PCW. a, a0,a00 PAX6 was expressed in the proliferative zone of LCGE but not

MCGE; PAX6 was also expressed in the VCGE with a distinct

cortical/subcortical boundary (arrow in a00). b, b0, b00 NKX2.1 was

largely confined to the caudal extension of MGE (MCGE) but with

some dispersion into the LCGE. c, c0, c00 Calretinin (CalR) was

preferentially expressed in the VZ and SVZ of the LCGE and only

scattered cells were observed in MCGE; CalR was also preferentially

expressed in VCGE with distinct pallial/subpallial boundary (arrow in

c00). Scale bars 1 mm in c (and for a, b) c 100 lm in c00 (and for a0, a00,b0, b00, c0). Ant anterior, Pos posterior


123

NKX2.1?/OLIG2-, and NKX2.1?/OLIG2? (Fig. 5a).

To confirm that OLIG2? cells at this stage of human

forebrain development (8–12 PCW) were GABAergic

interneuron precursors, we performed OLIG2 and GAD65/

67 double labelling, and found most of OLIG2? cells

expressed GAD65/67 (Fig. 5b, c). Furthermore, a propor-

tion of cells in the MGE were triple labelled with OLIG2,

NK2.1, and GAD65/67 (Fig. 5d–g). However, although

both OLIG2 and CalR were expressed in LCGE and MCGE,

no double labelling for these two markers was detected

(Suppl. Figure 3S).

Expression of OLIG2 in human fetal dorsal


At 8 PCW, a quite different pattern of OLIG2 expression

was observed in the posterior and anterior cortex. A stream

of OLIG2? cells from the GE appeared to be starting to

Fig. 3 Expression of NKX2.1 in the human fetal forebrain. a Double

labelling for GAD65/67 (green) and NKX2.1 (red) in coronal section

at 8 PCW. GAD65/67 was expressed mainly in the subventricular

zone (SVZ) of the MGE and LGE. In LGE, GAD65/67 immunore-

activity also showed a clear cortical/subcortical boundary anteriorly

(Ant; arrow). NKX2.1 was expressed in the ventricular zone (VZ) and

subventricular zone (SVZ) of the MGE, and in cells probably

migrating through the non-proliferative mantle zone of LGE toward

the cortex (crx). The high magnification inset in a shows NKX2.1/

GAD65/67 co-localisation in the cells of the SVZ only (NKX2.1 in

the nuclei, GAD65/67 in the cytoplasm). No NKX2.1? cells were

found in the cortex at 8 PCW. b–d At 12 PCW, the majority of cells

in the MGE were NKX2.1 immunoreactive; scattered NKX2.1? cells

were found in the proliferative zones of the LGE and cortex.

e Distribution of NKX2.1? cells in the proliferative zones of

different regions of human fetal forebrain at 12 PCW (see Table 2

for more details). In a, green vertical stripes in the cortex are an

artefact caused by section folding. Scale bars 1 mm in a and b,

200 lm in the inset for a; 100 lm in d (and for c)


123

invade the posterior cortex; however, no OLIG2? cells

were observed in the most posterior cortex at this stage

(Fig. 4c, d). In contrast, the anterior cortex was heavily

populated with strongly OLIG2 immunoreactive cells and

there was a moderate immunostaining throughout the cor-

tical wall including the cortical plate (Fig. 4c), as was

previously observed at 7.5 PCW (Al-Jaberi et al. 2015).

OLIG2 was also expressed in the VZ and SVZ of both the

MGE-like and the LGE-like septum, with a stream of

OLIG2? cells appearing to migrate from the septum into

the medial wall of the anterior cortex (Fig. 4c, e).

OLIG2? cells in the anterior cortex were double labelled

with KI67 showing that a significant number of

OLIG2? cells were dividing, which suggested either a

dorsal origin for these cells or that they retained prolifer-

ative capacity after migrating into the cortex (Fig. 6a).

At 12 PCW, a proportion of OLIG2? cells in the cortex

were also found to co-express KI67 (Fig. 6b). However,

OLIG2? cells were not double-labelled with either PAX6

or TBR2 (Fig. 6c, d), showing that OLIG2 is not expressed

by typical cortical radial glial progenitors or intermediate

progenitors (Bayatti et al. 2008a; Lui et al. 2011).

OLIG2? cells populated the whole cortex and were mainly

seen in the SVZ and IZ; nevertheless, scattered positive

cells were sometimes observed in the VZ and the cortical

plate (CP; Fig. 6e–i). We quantified the average density of

OLIG2? cells in the proliferative zones of the four dif-

ferent cortical regions. A higher density was found in the

medial cortex with a decreasing gradient to the latero-

ventral regions (Fig. 6k). Overall, OLIG2 expression was

far less confined to the MGE than NKX2.1, with approxi-

mately 38% of OLIG2? cells in proliferative layers found

in the MGE and ventral septum, 50% in the LGE and

VCGE, and 12% in the cortex (Fig. 6j; Table 2).

Expression of COUP-TFII in the ventral


In the ganglionic eminences at 8 PCW, COUPT-FTII was

largely confined to the CGE and the boundary between

MGE and LGE, although scattered cells were also observed

within the MGE and LGE (Fig. 7a–c). At 12 PCW, COUP-

TFII was mainly expressed in CGE compartments, mod-

erately expressed in LGE, with only scattered positive cells

found in the MGE (Fig. 7f–i). We quantified the average

density of COUP-TFII? cells in the proliferative zones of

MGE, septum, LGE, and VCGE (and cortex) in a coronally

cut brain at 12 PCW, and found that the proportion of

COUP-TFII? cells located in the VCGE (*39%) was

considerably higher than in the much larger LGE (27%)

with only a very small proportion found in the MGE and

ventral septum (\2%) (Fig. 7j; Table 2). However, strong

expression of COUP-TFII was observed in the VZ at the

boundary between the MGE and LGE (Fig. 7g). COUP-

TFII immunoreactivity also revealed a clear cortical/sub-

cortical boundary located ventral to the physical sulcus

between the cortex and the bulge of LGE (Fig. 7i).

Although COUP-TFII is expressed either side of the

boundary, there is markedly higher expression in the LGE.

Similarly, Pauly et al. (2014) reported an abrupt transition

from high to low DLX2 expression going from the LGE to

cortex in human at 7–8 PCW, even though PAX6 was

expressed on either side of the boundary (as we have

observed, see above).

The most dorsal LCGE showed high expression of

COUP-TFII but with no evidence of dividing (KI67

expressing) COUP-TFII? cells even in the proliferative

zones (Fig. 8a, b; Suppl. Figure 4a). Similarly, MCGE

showed relatively lower expression of COUP-TFII and in

post-mitotic cells only (Fig. 8a, b). Notably, the VCGE

showed strong expression of COUP-TFII in both the SVZ

and VZ, and most of COUP-TFII? cells in this region

were double labelled with the cell division marker KI67

(Fig. 8e; Suppl. Figure 4c) which is in agreement with

Hansen et al. (2013) who found a gradient of KI67 positive

COUP-TFII cells between the most ventral part of the CGE

and the more dorsal and anterior regions. Thus, the VCGE

is the birth place for all COUP-TFII? precursors in the

ganglionic eminences, but surprisingly most of these

COUP-TFII? precursors co-express PAX6, a marker for

dorsal radial glial progenitor cells (Suppl. Figure 4b and d).

We found most of the COUP-TFII? cells in the LCGE and

VCGE showed double labelling with CalR. However, there

were also a substantial number of cells that express these

two markers separately (Suppl. Figure 5a). The same

observations were also made in the LGE (Suppl. Fig-

ure 5b), suggesting that there is a distinct population of

CalR-expressing GABAergic interneurons which are

COUP-TFII independent.

Expression of COUP-TFII in the dorsal


We found a distinct distribution of COUP-TFII? cells

between the anterior and posterior cortex at 8 PCW. In the

anterior cortex, COUPT-FII protein was localized to all

layers. Although most of COUP-TFII? cells were located

in the SVZ and IZ, a considerable number of cells were

also observed in the VZ and CP (Fig. 7c, e). A different

distribution of COUP-TFII? cells was observed in the

posterior cortex, where cells were restricted to what

appeared to be two migratory streams; a major one in the

SVZ, and a less defined one in the nascent pre-subplate at

the border between the IZ and the CP (Fig. 7c, d). No

COUP-TFII positive cells were found in the VZ (Suppl.

Figure 5d).


123


123

By 12 PCW, we estimated that about 32% of all COUP-

TFII? cells in proliferative zones of the telencephalon were

located in the cortex (Table 2) and we observed particularly

dense immunoreactivity for COUP-TFII in the VZ and SVZ

of the ventral parts of the frontal and temporal cortex located

close to the VCGE (Suppl. Figure 4c and d; Fig. 9a, b). Most

of COUP-TFII? cells in the VZ of ventral cortex showed a

radial morphology, whereas cells in the VZ of the VCGE

showed disorganized morphology (Suppl. Figure 4c). Sim-

ilar to the VCGE, most of COUP-TFII? cells in the VZ of

ventral cortex were double labelled with KI67 (Suppl. Fig-

ure 4c). Furthermore, we also found double labelling of

COUP-TFII cells with the radial glial progenitor cell marker

PAX6 (Suppl. Figure 4d) and the post-mitotic glutamatergic

neuron marker TBR1 (Suppl. Figure 6b). However, in the

bFig. 4 Expression pattern of OLIG2 in human fetal forebrain at 8 and

12 PCW. a OLIG2 was strongly expressed in the proliferative zones

of MGE, weaker expression was observed in LGE. b Higher

magnification of boxed area in a. c Anterior (ant) cortex was heavily

populated with OLIG2? cells, whereas no OLIG2? cells were found

in the most posterior cortex. d Higher magnification of boxed area in

c, OLIG2? cells from the GE appeared to be only starting to invade

the posterior cortex (arrows). e OLIG2 was expressed in the

proliferative zone of septum, with a stream of OLIG2? cells

appearing to migrate (arrow) into the medial cortex (crx). f, g Similar

to 8 PCW, OLIG2 was strongly expressed in the proliferative zone of

MGE at 12 PCW, with aggregations of OLIG? cells amongst

OLIG2- cells. h Relatively weaker expression was observed in LGE.

i, j Expression pattern of OLIG2 in the MGE and LGE extended to

the MCGE and LCGE, respectively. k Scattered OLIG2? cells were

observed in the VCGE. In a dark vertical stripes in the cortex are an

artefact caused by section folding. Scale bars 1 mm in a, c, f, and i;100 lm in b, d, e, g, h, j, and k

Fig. 5 a Double labelling for NKX2.1 (green) and OLIG2 (red) in

the MGE at 8 PCW showed three populations of cells located in the

MGE: the first expressed only OLIG2 (red), the second expressed

only NKX2.1 (green), and a third population co-localized these two

markers (yellow). b, c Double labelling for OLIG2 (red) and GAD65/

67 (green) in the MGE at 8 and 12 PCW showed that most of the

OLIG2? cells (nuclear staining) were double labelled with GAD65/

67. Magnification inset in b shows double labelling in the SVZ but not

the VZ. d–g Triple labelling for NKX2.1 (green), OLIG2 (red), and

GAD65/67 (purple) in the MGE at 8 PCW showed that many cells co-

expressed the transcription factors NKX2.1 and OLIG2 (nuclear

staining, yellow) and GAD65/67 (cytoplasmic, purple). Scale bars

100 lm in a; 50 lm in b (15 lm in inset), c 20 lm in g (and for d–f)


123

dorsal cortex, although we observed a substantial number of

COUPT-FII? cells in the VZ and SVZ, no double labelling

with KI67 or co-expression with PAX6, TBR2 (not shown),

or TBR1 was observed (Suppl. Figure 6a).

All these findings suggest that the neurogenic domain for

COUPT-FII precursors in the VCGE extends into the cortical

wall of the ventral cortex, but not dorsal cortex, and includes

radial glial progenitor cells that generate glutamatergic neu-

rons. We quantified the average density of COUP-TFII? cells

in the proliferative zones across the cortex (Fig. 9a–f) and

found a decreasing gradient of density from higher gradient in

the ventral cortex to a lower gradient more dorsally. In a recent

study, Reinchisi et al. (2012) reported that COUP-TFII? cells

are more abundant in the temporal/caudal cortex of human

fetal brain, which was attributed to a caudal migratory stream

from the CGE. However, our results suggest, in humans, the

presence of an additional anterior migratory stream, and

provide evidence that the neurogenic domain of COUP-TFII

expressing progenitor cells is not confined to the CGE, but

extends to the ventral cortex, including the frontal lobe

(Fig. 9g). In addition, we found most COUP-TFII? cells in

the ventral cortex to be double-labelled with CalR. However, a

proportion of COUP-TFII? cells in all other regions of the

cortex were also shown to be a subpopulation of CalR

expressing cells (Suppl. Figure 5c–e).

Discussion

We have described the expression patterns of three tran-

scription factors important to the generation of cortical

interneurons in the early fetal human telencephalon and

Fig. 6 a Significant number of OLIG2? cells (red) in the anterior

cortex co-expressed the cell division marker KI67 (green) at 8 PCW

(arrows). b Many OLIG2? cells co-expressed KI67 at 12 PCW

(arrows). c OLIG2? cells (red) did not co-express the radial glial

progenitor cell marker PAX6 (green). d OLIG2? cells (red) did not

co-express the intermediate progenitor cell marker TBR2 (green).

e OLIG2 expression in the cortex (coronal section) at 12 PCW.

OLIG2 expression in ventral cortex (f), lateral cortex (g), dorsal

cortex (h), and medial cortex (i). j Distribution of OLIG2? cells in

the proliferative zones of different regions of human fetal forebrain at

12 PCW. k Average density of OLIG2? cells in the ventral cortex,

lateral cortex, dorsal cortex, and medial cortex of 12 PCW human

fetal brain. Scale bars 50 lm in a; 20 lm in b; 50 lm in c; 20 lm in

d; 1 mm in e; and 100 lm in i (and for g and h)


123

demonstrated that they occupy distinct (although overlap-

ping) neurogenic domains which can extend into the cor-

tex. NKX2.1 was very largely confined to the MGE,

MCGE, and ventral septum, and at this stage of develop-

ment, these observations support the previous studies

suggesting that interneuron generation from NKX2.1 pos-

itive cells may be identical in nature with the process

occurring in rodents (Hansen et al. 2013; Ma et al. 2013;

Arshad et al. 2016). OLIG2 was expressed by cells in the

proliferative zones of the MGE, MCGE, and septum, co-

expressed with GAD65/67, but not necessarily co-ex-

pressed with NKX2.1, and was also extensively expressed

in the LGE, LCGE, and in dividing cells in the cortex;

observations previously unreported at this key stage of

development. Within the ganglionic eminences, dividing

COUP-TFII precursors were localized to the VCGE as

previously described (Hansen et al. 2013) but were also

numerous in adjacent regions of ventral cortex. From

careful examination of multiple expression patterns, we

have been able to more accurately compartmentalise the

CGE than has previously been attempted, and describe

additional ventral to dorsal migratory streams for

interneuron precursors not previously reported in rodents,

as will be discussed in more detail below. Further evidence

for interneuron generation in the human dorsal telen-

cephalon has been presented.

Anatomical and molecular subdivisions of the CGE

In rodents, the CGE has been identified as a source of

specific GABAergic interneuronal subtypes different from

those generated from the MGE (Miyoshi et al. 2010; Rudy

et al. 2011). Some researchers have concluded that the

CGE comprises caudal extensions of LGE and MGE,

respectively, by virtue of its gene expression patterns

(Corbin et al. 2003; Flames et al. 2007). However, as

additional transcription factors, such as COUP-TFI and

COUP-TFII, are enriched in CGE, it is possible that the

CGE has evolved as a distinct neurogenic domain separate

from the MGE and LGE (Kanatani et al. 2008). This study

has revealed that in the developing human brain, the lateral

and medial portions of the CGE share the expression pat-

terns of PAX6, OLIG2, and NKX2.1 of the LGE and MGE,

respectively. However, in addition to these lateral and

medial portions of the CGE, the extension of the CGE

along the lateral ventricle into the greatly enlarged tem-

poral lobe has produced a third compartment distinguish-

able by its characteristic co-localisation of intense COUP-

TFII and PAX6 expression in the proliferative layers.

Dividing COUP-TFII? cells were confirmed as being

confined to this ventral region of the CGE (Hansen et al.,

2013). In addition, unlike the dorsally located lateral and

medial portions, almost no NKX2.1? cells were found in

the VCGE. These findings suggest that the anatomical and

molecular boundaries of the CGE should be defined care-

fully and separately, with the dorsal region formed from

caudal extensions of the LGE and MGE, albeit with a

higher density of post-mitotic COUP-TFII and CalR posi-

tive cells, and the ventral region having its own molecular

signature including COUP-TFII positive progenitor cells.

Are anterior and medial migratory streams

prominent in the human telencephalon?

Quantitative PCR, microarray, in situ hybridisation, and

immunohistochemical studies between 8–12 PCW have

previously identified an anterior-to-posterior gradient of

expression of multiple genes identified with GABAergic

interneurons and GABAergic neurotransmission, including

transcription factors characteristic of interneuron precur-

sors, isoforms of GAD, GABA receptor sub-units, and

calcium-binding proteins (Bayatti et al. 2008a; Ip et al.

2010; Al-Jaberi et al. 2015) seemingly at odds with the

accepted lateral (MGE derived) and posterior (CGE

derived) pathways of migration for interneuron precursors

from ventral to dorsal telencephalon (Wonders and

Anderson 2006). This led to speculation that the anterior

cortex in particular may be a novel site for generation of

interneurons in the primate telencephalon, perhaps, to

populate the enlarged prefrontal lobes of the primate brain

(Al-Jaberi et al. 2015; Clowry 2015). This study offers up

the alternative explanation that migrating interneurons may

more rapidly invade the anterior than the posterior cortex,

even from apparently caudal structures, such as the VCGE.

We saw evidence of a rostral migratory stream of COUP-

TFII and CalR expressing cells from the VCGE, where

COUPTFII expressing progenitors exclusively underwent

division, to the anterior cortex via the LCGE, LGE, and

ventral pallium (Fig. 9). Such cells were more numerous in

the anterior than posterior cortex, as previously described

for CalR? neurons (Bayatti et al. 2008a). Examination of

our 3D reconstructions of the 12 PCW fetal brain con-

firmed that this path length is similar or even shorter than

that from the VCGE to the dorso-posterior cortex via the

temporal lobe (Fig. 9g). Although rostral migratory

streams from the LGE to olfactory bulbs are well described

in mammals (Corbin et al. 2001; Waclaw et al. 2009), a

rostral migratory stream from the GE to the rostral pallium

has only been reported in shark (Quintana-Urzainqui et al.

2015) and so may be overlooked or missing in rodent

models.

In addition, we have observed increased expression of

OLIG2 in the anterior compared to posterior cortex in

agreement with the previous studies (Ip et al. 2010; Al-

Jaberi et al. 2015) particularly at 8 PCW where there was

also a distinct medial to lateral gradient of OLIG2


123


123

expression. In this case, the migratory stream appeared to

derive from progenitor cells in the MGE and sub-cortical

septum, and enter the cortex via the medial wall. This is in

direct contradiction to what has been reported in rodents

where interneurons populating medial wall-derived struc-

tures, such as the hippocampus, are described as deriving

from the MGE and CGE via lateral migration (Pleasure

et al. 2000; Wonders and Anderson 2006; Morozov et al.

2009; Faux et al. 2012). In our preparations, we found

evidence that OLIG2? and NKX2.1? progenitors reside

in the septum and OLIG2? cells, at least, migrate medially

to the cortex. Again, this is in disagreement with findings in

rodents, where septum derived cells were reported not to

enter the cortex at all (Rubin et al. 2010). Thus, we propose

that the human or primate brain possesses an additional

medial migratory pathway for GABAergic interneurons

populating frontal and medial areas of the cerebral cortex.

The much larger human cortex may require additional

migratory pathways compared to smaller mammalian

brains. However, it is worth noting that a medial migratory

pathway for Nkx2.1 positive precursors from the MGE to

the medial pallium has recently been reported in the shark

(Quintana-Urzainqui et al. 2015); therefore, such a path-

way cannot be proposed as evolutionarily novel to the

bFig. 7 Expression pattern of COUP-TFII in human fetal forebrain at

8 and 12 PCW. a, b At 8 PCW, COUP-TFII was mainly expressed in

the caudal part of the ganglionic eminences and at the boundary

between MGE and LGE. Scattered cells were also observed in MGE

and LGE. c Expression pattern of COUP-TFII in the anterior (ant) and

posterior (pos) cortex at 8 PCW. d Magnification of boxed area in the

posterior cortex in c; COUP-TFII expression appeared to be restricted

to two migratory streams, one in the subventricular zone (SVZ) and

one at the border between the intermediate zone (IZ) and the cortical

plate (CP). e Magnification of boxed area in the anterior cortex in

c COUP-TFII? cells was found in all layers of the cortex. f–h At 12

PCW, COUP-TFII was highly expressed in VCGE, moderately

expressed in LGE, with scattered cells found in the MGE. Strong

expression was observed in the VZ/SVZ at the boundary between

MGE and LGE (boxed area in g). i Distribution of COUP-TFII

expression showed a distinct cortical/subcortical boundary (arrow)

between LGE and cortex (crx). j The distribution of COUP-

TFII? cells in the proliferative zones of different regions of human

fetal forebrain at 12 PCW. Scale bars 1 mm a, c, f; 100 lm in b, d, e,

g, h, and i

Fig. 8 COUP-TFII was predominantly expressed in VCGE but

spanned sub-cortical/cortical domains in human fetal forebrain.

a COUP-TFII expression in a sagittal section at 12 PCW. b–

d COUP-TFII was highly expressed in the proliferative zone of LCGE

with lower expression in the LGE and anterior cortex (ant). e The

strongest expression was observed in the proliferative zone of VCGE

where COUP-TFII? cells were not organized radially. f Strong

expression of COUP-TFII in the VZ of ventral/temporal cortex with

radial nuclear morphology of COUP-TFII? cells. Scale bars 1 mm in

a; 100 lm in d (and for b and c); 100 lm in f (and for e). Dors dorsal,

Pos posterior, Temp temporal, Crx cortex


123

Fig. 9 a COUP-TFII expression in frontal cortex (coronal section, 12

PCW). b COUP-TFII expression in ventral cortex, c lateral cortex,

d dorsal cortex, and e the medial cortex. f Average density of COUP-

TFII? cells in the ventral, lateral, dorsal, and medial cortex.

g Schematic diagram showing the distribution of COUP-TFII? cells

in the telencephalon and proposed migratory paths from the VCGE to

the anterior and posterior cortex (large arrows). COUP-TFII

progenitors also underwent division in the ventral cortex, but the

migratory paths and phenotype of the cells remain unclear (small

arrows). Scale bars 1 mm in a, 100 lm in e (and for b–d). Ant

anterior cortex, Pos posterior cortex, Tem temporal cortex, Med

medial cortex, BG basal ganglia, ChP choroid plexus, MZ/CP

marginal zone/cortical plate, SP/IZ presubplate/intermediate zone,

SVZ/VZ subventricular zone/ventricular zone


123

human brain. Instead, we might speculate that this is

missing or relatively small and overlooked in rodent

compared to other vertebrate species.

Potential dorsal telencephalic origin of GABAergic

interneurons

Based on studies carried out principally around mid-ges-

tation, Radonjic et al. (2014a) proposed that three mecha-

nisms exist for the production of cortical interneurons in

primates: generation in the ventral telencephalon followed

by migration to the cortex, precursors arriving in the cortex

from the ventral telencephalon, and undergoing further

division intra-cortically, and cortically derived progenitors

giving rise to interneurons. The last two proposals are

controversial, being firmly rejected by recent influential

and persuasive studies (Hansen et al. 2013; Ma et al. 2013;

Arshad et al. 2016). However, our present study found

clear evidence for the second mechanism. OLIG2? pre-

cursors appeared to follow migratory paths into the cortex;

however, OLIG2? cells were also shown to be undergoing

proliferation and these OLIG2? cells did not co-express

any markers of cortically derived progenitors, such as

PAX6 or TBR2 (although such double-labelling has been

reported at later stages of human development; Jakovceski

and Zecevic 2005). This firmly suggests that OLIG2 is not

immediately downregulated in cells entering the cortex

from subcortical structures, unlike NKX2.1, and that these

cells may retain the ability to divide within the cortex,

preferentially within anterior and medial locations, where

the highest density of such cells was found. However, there

also remains the possibility that OLIG2?/TBR2- interme-

diate progenitor cells are generated by cortical radial glial

progenitor cells which go on to produce GABAergic

interneurons.

It is also clear that in the more ventral areas of the

anterior and temporal cortex, there is high expression of

COUP-TFII expressing progenitor cells and post-mitotic

neurons. These progenitor cells co-express either PAX6

or TBR2 and post-mitotic cells co-expressing TBR1 and

COUPTFII were also observed, which demonstrates that

in the cortex, dividing COUP-TFII? progenitors give

rise to glutamatergic neurons. Although there are also

COUP-TFII?/CalR? presumptive interneurons present,

it is impossible to judge whether these have migrated in

from the adjacent CGE, or been generated intra-corti-

cally. However, a neuronal progenitor marker GSX2,

expressed upstream of COUP-TFII, which localises to

the LGE and CGE in rodent (Hsieh-Li et al. 1995; Wang

et al. 2013), has been found to be expressed in cells

undergoing division in the VZ/SVZ of the human fetal

cortex (Radonjic et al. 2014b) making intra-cortical

generation a possibility.

Whether or not proliferative NKX2.1? progenitor cells

are present in the cortex is contentious. Our observation at

12 PCW of NKX2.1? cells throughout the latero-medial

extent of the cortical wall, making up about 2.4% of all

NKX2.1? cells in the proliferative zones of the telen-

cephalon at this time, is in conflict with Hansen et al.

(2013) who reported nearly no NKX2.1? cells in the

cortex and only close to LGE/lateral cortex border. How-

ever, our findings are in partial agreement with Radonjic

et al. (2014a) who found NKX2.1? cells in the cortical

wall of human and macaque monkey fetal forebrains (at

later stages of development, 15–22 PCW for human)

undergoing active division, as did Arshad et al. (2016) in

human between 16–28 PCW although in very small num-

bers. As no NKX2.1? cells were seen in the cortex at

8PCW in agreement with the previous studies (Hansen

et al. 2013; Pauly et al. 2014), we propose that with age,

the incidence of NKX2.1? cells in the cortex gradually

increases, along with the capacity to undergo proliferation.

Whether these cells are generated in the cortex or have

migrated there from the ventral telencephalon without

downregulating NKX2.1 remains a question for further

investigation.

OLIG2 and COUP-TFII as regulators of cortical

arealisation

The division of the cerebral cortex into functional areas

(the cortical map) differs little between individuals in any

given species (Rakic et al. 2009). The previous work on

rodent development has identified certain transcription

factors (e.g., PAX6, SP8, EMX2, and COUP-TFI)

expressed in gradients across the neocortex that appear to

control regional expression of cell adhesion molecules and

organization of area specific thalamocortical afferent pro-

jections (Lopez-Bendito and Molnar 2003; O’Leary et al.

2007; Rakic et al. 2009). There may be common mecha-

nisms between species, as the developing human neocortex

displays counter-gradients of PAX6 and EMX2 at the early

stages of cortical development (Bayatti et al. 2008b).

However, the human cerebral cortex is composed of dif-

ferent and more complex local area identities and so might

be specified by a wider range of transcription factor gra-

dients; for instance, an anterior-to-posterior gradient of

CTIP2 expression has been observed in human early fetal

cortex (Ip et al. 2011). In this study, we observed a

prominent anterior-to-posterior gradient of OLIG2

expression, and a ventral-to-dorsal gradient of COUP-TFII

expression. In both the cases, the transcription factors are

also expressed at moderate levels in the cortical plate as

well as the proliferative zones, suggesting that areal spec-

ification mechanisms in cells extend into the post-mitotic

period. The extent to which these gradients interact with


123

interneuron precursors is not known, but we might specu-

late that OLIG2 or COUP-TFII controls expression of cell

adhesion molecules locally that attract migrating cells

expressing the same transcription factors, setting up the

migratory pathways into the cortex for interneurons arriv-

ing medially via the septum (OLIG2?) or laterally via

ventral anterior or temporal cortex (COUP- TFII?).

Conclusion

Evidence continues to accumulate that cortical GABAergic

interneuron production in primates differs in certain details

from what has been learnt from our rodent models. A

higher proportion of interneurons arise from the CGE in

primates and we provide a description of the compart-

mentalisation of the CGE. This study presents further

evidence that interneuron precursor cells may undergo

division in the cortex, although it remains to be proven

whether they are originally generated in the dorsal telen-

cephalon. Finally, whereas in rodents, interneuron precur-

sors are believed to enter the cortex from the ganglionic

eminences exclusively via lateral and posterior routes, in

human, we provide evidence of pathways via the anterior

and medial cortex.

Acknowledgements The human embryonic and fetal material was

provided by the jointly funded Human Developmental Biology

Resource (http://www.hdbr.org) MRC/Wellcome Trust grant#

099175/Z/12/Z). Ayman Alzu’bi is in receipt of a studentship from

Yarmouk University, Jordan. We are grateful to Dr Peter Thelwall for

capturing the MRI scans and Xin Xu for her help with slide scanning.

Open Access This article is distributed under the terms of the

Creative Commons Attribution 4.0 International License (http://crea

tivecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided you give

appropriate credit to the original author(s) and the source, provide a

link to the Creative Commons license, and indicate if changes were

made.

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